Fig 1: Schematic depiction of the proposed role of PDI in mediating chemically induced ferroptosis.
Fig 2: Model for the trafficking of vRNPs across the cytoplasm in an IAV-infected cell. The remodeled tubulo-vesicular ER, positive for the PDI marker (in blue) extends around the MTOC (in gray) and from the nuclear envelope (ne) to the plasma membrane (pm). After their exit from the nucleus, individual vRNPs and/or sub-bundles of vRNPs are targeted to the modified ER. ICVs loaded with vRNPs and with the Rab11 molecule (in red) might bud from the ER and ensure the transport of vRNPs to the plasma membrane. The frequently observed pairing of ICVs could favor RNA−RNA interactions among vRNPs and the progressive assembly of sets of 8 distinct vRNPs. vRNPs are released from ICVs and possibly transferred to the plasma membrane in a touch-and-go process
Fig 3: Localization of Rab11 and PDI in WSN-infected A549 cells. a, b Immunofluorescence and confocal microscopy images of two A549 cells (one optical section per cell) infected with the WSN virus and labeled at 8 hpi with antibodies specific for Rab11 (red) and the ER marker PDI (green). c, d Two optical sections of a control mock-infected cells. Arrows point to co-localization spots. Scale bar, 5 μm
Fig 4: PDI contributed to erastin-induced lipid ROS accumulation. (A to D) Effect of erastin on PDI protein and mRNA levels in MDA-MB-231 and 786-O cells. Protein levels of PDI were measured using Western blot analysis (A, B), and its mRNA levels were analyzed using qRT-PCR (C, D). Data are means ± SD (n = 3). **, P < 0.01; n.s., not significant. (E to G) PDI knockdown by siRNAs reduced ROS levels. After transfection with siRNAs for 48 h, ROS levels were assessed following DCFH-DA (E) staining coupled with flow cytometry analysis (F, G). Images were taken with a fluorescence microscope (E). Scale bar = 50 μm. Data are means ± SD (n = 3). ****, P < 0.0001; n.s., not significant. (H) Knockdown of PDI by shRNAs was confirmed by Western blotting. (I) Knockdown of PDI with shRNAs reduced lipid ROS levels, as assessed by C11-BODIPY staining. Scale bar = 20 μm.
Fig 5: PDI was involved in iNOS dimerization. (A) Confirmation by Western blotting of PDI knockdown by siRNAs. Three individual PDI siRNAs were mixed and transfected at a working concentration of 100 nM with the RNAiMAX reagent. (B) PDI knockdown by siRNAs (48 h) significantly suppressed iNOS dimerization in MDA-MB-231 and 786-O cells treated with erastin. (C) Lysates from iNOS-overexpressing HEK-293 cells were incubated with or without purified PDI protein (1 mg/mL) at 4°C for 1 h, and then the dimer and monomer forms of iNOS were determined by nondenaturing SDS-PAGE (with 30 μg protein in each lane). (D, E) Dimer and monomer forms of iNOS were detected after incubation of purified iNOS protein (5 μg) with or without PDI (1 mg/mL) at 4°C for 1 h (C) or in the presence of 100 μM cystamine at 4°C for 1 h (E). The ratios represent the intensity of bands of dimer or monomer forms of iNOS normalized against the vehicle control group. Representative experiments in panels A to E were repeated at least three times with similar results. (F to H) Reduction in NO or lipid ROS levels was detected in 5 μM erastin-treated cells in the presence of 100 μM cystamine. After treatment for 8 h, accumulation of NO and ROS was determined following DAF-FM-DA (F, G) or C11-BODIPY (H) labeling coupled with flow cytometry. Scale bar = 20 μm. (F) Images are representative of three independent experiments.
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